Cell Entry Pathway Research Articles

Recreating the biological steps of viral infection on a cell-free bioelectronic platform to profile viral variants of concern

Viral mutations frequently outpace technologies used to detect harmful variants. Given the continual emergence of SARS-CoV-2 variants, platforms that can identify the presence of a virus and its propensity for infection are needed. Our electronic biomembrane sensing platform recreates distinct SARS-CoV-2 host cell entry pathways and reports the progression of entry as electrical signals. We focus on two necessary entry processes mediated by the viral Spike protein: virus binding and membrane fusion, which can be distinguished electrically. We find that closely related variants of concern exhibit distinct fusion signatures that correlate with trends in cell-based infectivity assays, allowing us to report quantitative differences in their fusion characteristics and hence their infectivity potentials. We use SARS-CoV-2 as our prototype, but we anticipate that this platform can extend to other enveloped viruses and cell lines to quantifiably assess virus entry.

Evaluation of potency and metabolic stability of diphyllin-derived Vacuolar-ATPase inhibitors

Diphyllin is a naturally occurring lignan comprised of an aryl naphthalene lactone scaffold that demonstrates beneficial biological activities in disease models of cancer, obesity, and viral infection. A target of diphyllin and naturally occurring derivatives is the vacuolar ATPase (V-ATPase) complex. Although diphyllin-related natural products are active with in vitro models for viral entry, the potencies and unknown pharmacokinetic properties limit well-designed in vivo evaluations. Previous studies demonstrated that diphyllin derivatives have the utility of blocking the Ebola virus cell entry pathway. However, diphyllin shows limited potency and poor oral bioavailability in mice. An avenue to improve the potency was used in a new library of synthetic derivatives of diphyllin. Diphyllin derivatives exploiting ether linkages at the 4-position with one-to-three carbon spacers to an oxygen or nitrogen atom provided compounds with EC50 values ranging from 7 to 600 nM potency and selectivity up to >500 against Ebola virus in infection assays. These relative potencies are reflected in the Ebola virus infection of primary macrophages, a cell type involved in early pathogenesis. A target engagement study reveals that reducing the ATPV0a2 protein expression enhanced the potency of diphyllin derivatives to block EBOV entry, consistent with effects on the endosomal V-ATPase function. Despite the substantial enhancement of antiviral potencies, limitations were identified, including rapid clearance predicted by in vitro microsome stability assays. However, compounds with similar or improved half-lives relative to diphyllin demonstrated improved pharmacokinetic profiles in vivo. Importantly, these derivatives displayed suitable plasma levels using oral administration, establishing the feasibility of in vivo antiviral testing.

PH-dependent interactions of coacervate-forming histidine-rich peptide with model lipid membranes

Peptide-based liquid droplets (coacervates) produced by spontaneous liquid-liquid phase separation (LLPS), have emerged as a promising class of drug delivery systems due to their high entrapping efficiency and the simplicity of their formulation. However, the detailed mechanisms governing their interaction with cell membranes and cellular uptake remain poorly understood. In this study, we investigated the interactions of peptide coacervates composed of HBpep—peptide derived from the histidine-rich beak proteins (HBPs) of the Humboldt squid—with model cellular membranes in the form of supported lipid bilayers (SLBs). We employed quartz crystal microbalance with dissipation monitoring (QCM-D), neutron reflectometry (NR) and atomistic molecular dynamics (MD) simulations to reveal the nature of these interactions in the absence of fluorescent labels or tags. HBpep forms small oligomers at pH 6 whereas it forms µm-sized coacervates at physiological pH. Our findings reveal that both HBpep oligomers and HBpep-coacervates adsorb onto SLBs at pH 6 and 7.4, respectively. At pH 6, when the peptide carries a net positive charge, HBpep oligomers insert into the SLB, facilitated by the peptide’s interactions with the charged lipids and cholesterol. Importantly, however, HBpep coacervate adsorption at physiological pH, when it is largely uncharged, is fully reversible, suggesting no significant lipid bilayer rearrangement. HBpep coacervates, previously identified as efficient drug delivery vehicles, do not interact with the lipid membrane in the same manner as traditional cationic drug delivery systems or cell-penetrating peptides. Based on our findings, HBpep coacervates at physiological pH cannot cross the cell membrane by a simple passive mechanism and are thus likely to adopt a non-canonical cell entry pathway.

Programming an Enhanced Uptake and the Intracellular Fate of Magnetic Microbeads.

This study compares two kinds of magnetic microbeads with different surface features and cell entry pathways, aiming to provide insights into how to program their cell uptake and intracellular fate. It is found that a rougher surface enhances the cell uptake of the microbeads, regardless of whether they are pulled by a magnetic field gradient or adsorbed by the cell membrane. However, the entry route affects the intracellular localization of the microbeads: The magnetically dragged microbeads reach the cytoplasm, while the adsorbed microbeads stay in the late endosomes and lysosomes. This suggests that different strategies can be used to target different cellular compartments with magnetic microbeads. Moreover, it is demonstrated that the cells containing the microbeads can be moved and regrown at specific locations by applying a magnetic field gradient, showing the potential of these magnetic microbeads for cell delivery and manipulation.

Amyloid-Beta Peptides 40 and 42 Employ Distinct Molecular Pathways for Cell Entry and Intracellular Transit at the Blood-Brain Barrier Endothelium.

The blood-brain barrier (BBB) plays a critical role in maintaining the equilibrium between amyloid beta (Aβ) levels in blood and the brain by regulating Aβ transport. Our previous publications demonstrated that BBB trafficking of Aβ42 and Aβ40 is distinct and is disrupted under various pathophysiological conditions. However, the intracellular mechanisms that allow BBB endothelium to differentially handle Aβ40 and Aβ42 have not been clearly elucidated. In this study, we identified mechanisms of Aβ endocytosis in polarized human cerebral microvascular endothelial cell monolayers. Our studies demonstrated that Aβ peptides with fluorescent label (F-Aβ) were internalized by BBB endothelial cells via energy, dynamin, and actin-dependent endocytosis. Interestingly, endocytosis of F-Aβ40 but not F-Aβ42 was substantially reduced by clathrin inhibition, whereas F-Aβ42 but not F-Aβ40 endocytosis was reduced by half after inhibiting the caveolae-mediated pathway. Following endocytosis, both isoforms were sorted by the endo-lysosomal system. Although Aβ42 was shown to accumulate more in the lysosomes, which could lead to its higher degradation and/or aggregation at lower lysosomal pH, Aβ40 demonstrated robust accumulation in recycling endosomes, which may facilitate its exocytosis by the endothelial cells. These results provide a mechanistic insight into the selective ability of BBB endothelium to transport Aβ40 versus Aβ42. This knowledge contributes to the understanding of molecular pathways underlying Aβ accumulation in the BBB endothelium and associated BBB dysfunction. Moreover, it allows us to establish mechanistic rationale for altered Aβ40:Aβ42 ratios and anomalous amyloid deposition in the cerebral vasculature as well as brain parenchyma during Alzheimer's disease progression. SIGNIFICANCE STATEMENT: Differential interaction of Aβ40 and Aβ42 isoforms with the blood-brain barrier (BBB) endothelium may contribute to perturbation in Aβ42:Aβ40 ratio, which is associated with Alzheimer's disease (AD) progression and severity. The current study identified distinct molecular pathways by which Aβ40 and Aβ42 are trafficked at the BBB, which regulates equilibrium between blood and brain Aβ levels. These findings provide molecular insights into mechanisms that engender BBB dysfunction and promote Aβ accumulation in AD brain.

Comprehensive biophysical characterization of AAV-AAVR interaction uncovers serotype- and pH-dependent interaction

After more than two decades of research and development, adeno-associated virus (AAV) has become one of the dominant delivery vectors in gene therapy. Despite the focused research, the cell entry pathway for AAV is still not fully understood. Universal AAV receptor (AAVR) has been identified to be involved in cellular entry of different AAV serotypes. With the unveiling of the high-resolution AAV-AAVR complex structure by cryogenic electron microscopy, the atomic level interaction between AAV and AAVR has become the focus of study in recent years. However, the serotype dependence of this binding interaction and the effect of pH have not been studied. Here, orthogonal approaches including bio-layer interferometry (BLI), size-exclusion chromatography coupled to multi-angle laser scattering (SEC-MALS) and sedimentation velocity analytical ultracentrifugation (SV-AUC) were utilized to study the interaction between selected AAV serotypes and AAVR under different pH conditions. A robust BLI method was developed and the equilibrium dissociation binding constants (KD) between different AAV serotypes (AAV1, AAV5 and AAV8) and AAVR was measured. The binding constants measured by BLI together with orthogonal methods (SEC-MALS and SV-AUC) all confirmed that AAV5 has the strongest binding affinity followed by AAV1 while AAV8 binds the weakest. It was also observed that lower pH promotes the binding between AAV and AAVR and neutral or slightly basic conditions lead to very weak binding. These data indicate that for certain serotypes, AAVR may play a prominent role in trafficking AAV to the Golgi rather than acting as a host cell receptor. Information obtained from these combinatorial biophysical methods can be used to engineer future generations of AAVs to have better transduction efficiency.

Adaptive variations in SARS-CoV-2 spike proteins: effects on distinct virus-cell entry stages.

Evolved SARS-CoV-2 variants of concern (VOCs) spread through human populations in succession. Major virus variations are in the entry-facilitating viral spike (S) proteins; Omicron VOCs have 29-40 S mutations relative to ancestral D614G viruses. The impacts of this Omicron divergence on S protein structure, antigenicity, cell entry pathways, and pathogenicity have been extensively evaluated, yet gaps remain in correlating specific alterations with S protein functions. In this study, we compared the functions of ancestral D614G and Omicron VOCs using cell-free assays that can reveal differences in several distinct steps of the S-directed virus entry process. Relative to ancestral D614G, Omicron BA.1 S proteins were hypersensitized to receptor activation, to conversion into intermediate conformational states, and to membrane fusion-activating proteases. We identified mutations conferring these changes in S protein character by evaluating domain-exchanged D614G/Omicron recombinants in the cell-free assays. Each of the three functional alterations was mapped to specific S protein domains, with the recombinants providing insights on inter-domain interactions that fine-tune S-directed virus entry. Our results provide a structure-function atlas of the S protein variations that may promote the transmissibility and infectivity of current and future SARS-CoV-2 VOCs. IMPORTANCE Continuous SARS-CoV-2 adaptations generate increasingly transmissible variants. These succeeding variants show ever-increasing evasion of suppressive antibodies and host factors, as well as increasing invasion of susceptible host cells. Here, we evaluated the adaptations enhancing invasion. We used reductionist cell-free assays to compare the entry steps of ancestral (D614G) and Omicron (BA.1) variants. Relative to D614G, Omicron entry was distinguished by heightened responsiveness to entry-facilitating receptors and proteases and by enhanced formation of intermediate states that execute virus-cell membrane fusion. We found that these Omicron-specific characteristics arose from mutations in specific S protein domains and subdomains. The results reveal the inter-domain networks controlling S protein dynamics and efficiencies of entry steps, and they offer insights on the evolution of SARS-CoV-2 variants that arise and ultimately dominate infections worldwide.

SARS-CoV-2 Infection of Human Neurons Is TMPRSS2 Independent, Requires Endosomal Cell Entry, and Can Be Blocked by Inhibitors of Host Phosphoinositol-5 Kinase.

2019 coronavirus disease (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition to respiratory illness, COVID-19 patients exhibit neurological symptoms lasting from weeks to months (long COVID). It is unclear whether these neurological manifestations are due to an infection of brain cells. We found that a small fraction of human induced pluripotent stem cell (iPSC)-derived neurons, but not astrocytes, were naturally susceptible to SARS-CoV-2. Based on the inhibitory effect of blocking antibodies, the infection seemed to depend on the receptor angiotensin-converting enzyme 2 (ACE2), despite very low levels of its expression in neurons. The presence of double-stranded RNA in the cytoplasm (the hallmark of viral replication), abundant synthesis of viral late genes localized throughout infected cells, and an increase in the level of viral RNA in the culture medium (viral release) within the first 48 h of infection suggested that the infection was productive. Productive entry of SARS-CoV-2 requires the fusion of the viral and cellular membranes, which results in the delivery of the viral genome into the cytoplasm of the target cell. The fusion is triggered by proteolytic cleavage of the viral surface spike protein, which can occur at the plasma membrane or from endosomes or lysosomes. We found that SARS-CoV-2 infection of human neurons was insensitive to nafamostat and camostat, which inhibit cellular serine proteases, including transmembrane serine protease 2 (TMPRSS2). Inhibition of cathepsin L also did not significantly block infection. In contrast, the neuronal infection was blocked by apilimod, an inhibitor of phosphatidyl-inositol 5 kinase (PIK5K), which regulates early to late endosome maturation. IMPORTANCE COVID-19 is a disease caused by the coronavirus SARS-CoV-2. Millions of patients display neurological symptoms, including headache, impairment of memory, seizures, and encephalopathy, as well as anatomical abnormalities, such as changes in brain morphology. SARS-CoV-2 infection of the human brain has been documented, but it is unclear whether the observed neurological symptoms are linked to direct brain infection. The mechanism of virus entry into neurons has also not been characterized. Here, we investigated SARS-CoV-2 infection by using a human iPSC-derived neural cell model and found that a small fraction of cortical-like neurons was naturally susceptible to infection. The productive infection was ACE2 dependent and TMPRSS2 independent. We also found that the virus used the late endosomal and lysosomal pathway for cell entry and that the infection could be blocked by apilimod, an inhibitor of cellular PIK5K.

SARS-CoV-2 Transplacental Transmission: A Rare Occurrence? An Overview of the Protective Role of the Placenta

The outbreak of the coronavirus disease 2019 (COVID-19) pandemic, caused by novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in a global public health crisis, causing substantial concern especially to the pregnant population. Pregnant women infected with SARS-CoV-2 are at greater risk of devastating pregnancy complications such as premature delivery and stillbirth. Irrespective of the emerging reported cases of neonatal COVID-19, reassuringly, confirmatory evidence of vertical transmission is still lacking. The protective role of the placenta in limiting in utero spread of virus to the developing fetus is intriguing. The short- and long-term impact of maternal COVID-19 infection in the newborn remains an unresolved question. In this review, we explore the recent evidence of SARS-CoV-2 vertical transmission, cell-entry pathways, placental responses towards SARS-CoV-2 infection, and its potential effects on the offspring. We further discuss how the placenta serves as a defensive front against SARS-CoV-2 by exerting various cellular and molecular defense pathways. A better understanding of the placental barrier, immune defense, and modulation strategies involved in restricting transplacental transmission may provide valuable insights for future development of antiviral and immunomodulatory therapies to improve pregnancy outcomes.

Protease-Responsive Potential-Tunable AIEgens for Cell Selective Imaging of TMPRSS2 and Accurate Inhibitor Screening.

Transmembrane protease serine 2 (TMPRSS2) is a plasma membrane protease that activates both spike protein of coronaviruses for cell entry and oncogenic signaling pathways for tumor progression. TMPRSS2 inhibition can reduce cancer invasion and metastasis and partially prevent the entry of SARS-CoV-2 into host cells. Thus, there is an urgent need for both TMPRSS2-selective imaging and precise screening of TMPRSS2 inhibitors. Here, we report a TMPRSS2-responsive surface-potential-tunable peptide-conjugated probe (EGTP) with aggregation-induced emission (AIE) features for TMPRSS2 selective imaging and accurate inhibitor screening. The amphiphilic EGTP was constructed with tunable surface potential and responsive efficiency with TMPRSS2 and its inhibitor. The rational construction of AIE luminogens (AIEgens) with modular peptides indicated that the cleavage of EGTP led to a gradual aggregation with bright fluorescence in high TMPRSS2-expressing cells. This strategy may have value for selective detection of cancer cells, SARS-CoV-2-target cells, and screening of protease inhibitors.

The role of invariant surface glycoprotein 75 in xenobiotic acquisition by African trypanosomes.

The surface proteins of parasitic protozoa mediate functions essential to survival within a host, including nutrient accumulation, environmental sensing and immune evasion. Several receptors involved in nutrient uptake and defence from the innate immune response have been described in African trypanosomes and, together with antigenic variation, contribute towards persistence within vertebrate hosts. Significantly, a superfamily of invariant surface glycoproteins (ISGs) populates the trypanosome surface, one of which, ISG75, is implicated in uptake of the century-old drug suramin. By CRISPR/Cas9 knockout and biophysical analysis, we show here that ISG75 directly binds suramin and mediates uptake of additional naphthol-related compounds, making ISG75 a conduit for entry of at least one structural class of trypanocidal compounds. However, ISG75 null cells present only modest attenuation of suramin sensitivity, have unaltered viability in vivo and in vitro and no alteration to suramin-invoked proteome responses. While ISG75 is demonstrated as a valid suramin cell entry pathway, we suggest the presence of additional mechanisms for suramin accumulation, further demonstrating the complexity of trypanosomatid drug interactions and potential for evolution of resistance.

ACE2-Independent Alternative Receptors for SARS-CoV-2.

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is highly contagious and remains a major public health challenge despite the availability of effective vaccines. SARS-CoV-2 enters cells through the binding of its spike receptor-binding domain (RBD) to the human angiotensin-converting enzyme 2 (ACE2) receptor in concert with accessory receptors/molecules that facilitate viral attachment, internalization, and fusion. Although ACE2 plays a critical role in SARS-CoV-2 replication, its expression profiles are not completely associated with infection patterns, immune responses, and clinical manifestations. Additionally, SARS-CoV-2 infects cells that lack ACE2, and the infection is resistant to monoclonal antibodies against spike RBD in vitro, indicating that some human cells possess ACE2-independent alternative receptors, which can mediate SARS-CoV-2 entry. Here, we discuss these alternative receptors and their interactions with SARS-CoV-2 components for ACE2-independent viral entry. These receptors include CD147, AXL, CD209L/L-SIGN/CLEC4M, CD209/DC-SIGN/CLEC4L, CLEC4G/LSECtin, ASGR1/CLEC4H1, LDLRAD3, TMEM30A, and KREMEN1. Most of these receptors are known to be involved in the entry of other viruses and to modulate cellular functions and immune responses. The SARS-CoV-2 omicron variant exhibits altered cell tropism and an associated change in the cell entry pathway, indicating that emerging variants may use alternative receptors to escape the immune pressure against ACE2-dependent viral entry provided by vaccination against RBD. Understanding the role of ACE2-independent alternative receptors in SARS-CoV-2 viral entry and pathogenesis may provide avenues for the prevention of infection by SARS-CoV-2 variants and for the treatment of COVID-19.

The P681H Mutation in the Spike Glycoprotein of the Alpha Variant of SARS-CoV-2 Escapes IFITM Restriction and Is Necessary for Type I Interferon Resistance.

The appearance of new dominant variants of concern (VOC) of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) threatens the global response to the coronavirus disease 2019 (COVID-19) pandemic. Of these, the alpha variant (also known as B.1.1.7), which appeared initially in the United Kingdom, became the dominant variant in much of Europe and North America in the first half of 2021. The spike (S) glycoprotein of alpha acquired seven mutations and two deletions compared to the ancestral virus, including the P681H mutation adjacent to the polybasic cleavage site, which has been suggested to enhance S cleavage. Here, we show that the alpha spike protein confers a level of resistance to beta interferon (IFN-β) in human lung epithelial cells. This correlates with resistance to an entry restriction mediated by interferon-induced transmembrane protein 2 (IFITM2) and a pronounced infection enhancement by IFITM3. Furthermore, the P681H mutation is essential for resistance to IFN-β and context-dependent resistance to IFITMs in the alpha S. P681H reduces dependence on endosomal cathepsins, consistent with enhanced cell surface entry. However, reversion of H681 does not reduce cleaved spike incorporation into particles, indicating that it exerts its effect on entry and IFN-β downstream of furin cleavage. Overall, we suggest that, in addition to adaptive immune escape, mutations associated with VOC may well also confer a replication and/or transmission advantage through adaptation to resist innate immune mechanisms. IMPORTANCE Accumulating evidence suggests that variants of concern (VOC) of SARS-CoV-2 evolve to evade the human immune response, with much interest focused on mutations in the spike protein that escape from antibodies. However, resistance to the innate immune response is essential for efficient viral replication and transmission. Here, we show that the alpha (B.1.1.7) VOC of SARS-CoV-2 is substantially more resistant to type I interferons than the parental Wuhan-like virus. This correlates with resistance to the antiviral protein IFITM2 and enhancement by its paralogue IFITM3. The key determinant of this is a proline-to-histidine change at position 681 in S adjacent to the furin cleavage site, which in the context of the alpha spike modulates cell entry pathways of SARS-CoV-2. Reversion of the mutation is sufficient to restore interferon and IFITM2 sensitivity, highlighting the dynamic nature of the SARS CoV-2 as it adapts to both innate and adaptive immunity in the humans.

Human Adenovirus Type 26 Infection Mediated by αvβ3 Integrin Is Caveolin-1-Dependent

ABSTRACTHuman adenovirus type 26 (HAdV26) has been recognized as a promising platform for vaccine vector development, and very recently vaccine against COVID-19 based on HAdV26 was authorized for emergency use. Nevertheless, basic biology of this virus, namely, pathway which HAdV26 uses to enter the cell, is still insufficiently known. We have shown here that HAdV26 infection of human epithelial cells expressing low amount of αvβ3 integrin involves clathrin and is caveolin-1-independent, while HAdV26 infection of cells with high amount of αvβ3 integrin does not involve clathrin but is caveolin-1-dependent. Thus, this study demonstrates that caveolin-1 is limiting factor in αvβ3 integrin-mediated HAdV26 infection. Regardless of αvβ3 integrin expression, HAdV26 infection involves dynamin-2. Our data provide for the first-time description of HAdV26 cell entry pathway, hence increase our knowledge of HAdV26 infection. Knowing that functionality of adenovirus vector is influenced by its cell entry pathway and intracellular trafficking, our results will contribute to better understanding of HAdV26 immunogenicity and antigen presentation when used as vaccine vector.IMPORTANCE In order to fulfill its role as a vector, adenovirus needs to successfully deliver its DNA genome to the host nucleus, a process highly influenced by adenovirus intracellular translocation. Thus, cell entry pathway and intracellular trafficking determine functionality of human adenovirus-based vectors. Endocytosis of HAdV26, currently extensively studied as a vaccine vector, has not been described so far. We present here that HAdV26 infection of human epithelial cells with high expression of αvβ3 integrin, one of the putative HAdV26 receptors, is caveolin-1- and partially dynamin-2-dependent. Since caveolin containing domains provide a unique environment for specific signaling events and participate in inflammatory signaling one can imagine that directing HAdV26 cell entry toward caveolin-1-mediate pathway might play role in immunogenicity of this virus. Therefore, our results contribute to better understanding of HAdV26 infection pathway, hence, can be helpful in explaining induction of immune response and antigen presentation by HAdV26-based vaccine vector.

SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway

Vaccines based on the spike protein of SARS-CoV-2 are a cornerstone of the public health response to COVID-19. The emergence of hypermutated, increasingly transmissible variants of concern (VOCs) threaten this strategy. Omicron (B.1.1.529), the fifth VOC to be described, harbours multiple amino acid mutations in spike, half of which lie within the receptor-binding domain. Here we demonstrate substantial evasion of neutralization by Omicron BA.1 and BA.2 variants in vitro using sera from individuals vaccinated with ChAdOx1, BNT162b2 and mRNA-1273. These data were mirrored by a substantial reduction in real-world vaccine effectiveness that was partially restored by booster vaccination. The Omicron variants BA.1 and BA.2 did not induce cell syncytia in vitro and favoured a TMPRSS2-independent endosomal entry pathway, these phenotypes mapping to distinct regions of the spike protein. Impaired cell fusion was determined by the receptor-binding domain, while endosomal entry mapped to the S2 domain. Such marked changes in antigenicity and replicative biology may underlie the rapid global spread and altered pathogenicity of the Omicron variant.

COVID-19: Omicron - the latest, the least virulent, but probably not the last variant of concern of SARS-CoV-2.

SummaryThe Omicron variant rapidly became the dominant SARS‐CoV‐2 strain in South Africa and elsewhere. This review explores whether this rise was due to an increased transmission of the variant or its escape from population immunity by an extensively mutated spike protein. The mutations affected the structure of the spike protein leading to the loss of neutralization by most, but not all, therapeutic monoclonal antibodies. Omicron also shows substantial immune escape from serum antibodies in convalescent patients and vaccinees. A booster immunization increased, however, the titre and breadth of antiviral antibody response. The cellular immune response against Omicron was largely preserved explaining a satisfying protection of boosted vaccinees against severe infections. Clinicians observed less severe infection with Omicron, but other scientists warned that this must not necessarily reflect less intrinsic virulence. However, in animal experiments with mice and hamsters, Omicron infections also displayed a lesser virulence than previous VOCs and lung functions were less compromised. Cell biologists demonstrated that Omicron differs from Delta by preferring the endocytic pathway for cell entry over fusion with the plasma membrane which might explain Omicron’s distinct replication along the respiratory tract compared with Delta. Omicron represents a distinct evolutionary lineage that deviated from the mainstream of evolving SARS‐CoV‐2 already in mid‐2020 raising questions about where it circulated before getting widespread in December 2021. The role of Omicron for the future trajectory of the COVID‐19 pandemic is discussed.

Virus-based vaccine vectors with distinct replication mechanisms differentially infect and activate dendritic cells

The precise mechanism by which many virus-based vectors activate immune responses remains unknown. Dendritic cells (DCs) play key roles in priming T cell responses and controlling virus replication, but their functions in generating protective immunity following vaccination with viral vectors are not always well understood. We hypothesized that highly immunogenic viral vectors with identical cell entry pathways but unique replication mechanisms differentially infect and activate DCs to promote antigen presentation and activation of distinctive antigen-specific T cell responses. To evaluate differences in replication mechanisms, we utilized a rhabdovirus vector (vesicular stomatitis virus; VSV) and an alphavirus-rhabdovirus hybrid vector (virus-like vesicles; VLV), which replicates like an alphavirus but enters the cell via the VSV glycoprotein. We found that while virus replication promotes CD8+ T cell activation by VLV, replication is absolutely required for VSV-induced responses. DC subtypes were differentially infected in vitro with VSV and VLV, and displayed differences in activation following infection that were dependent on vector replication but were independent of interferon receptor signaling. Additionally, the ability of the alphavirus-based vector to generate functional CD8+ T cells in the absence of replication relied on cDC1 cells. These results highlight the differential activation of DCs following infection with unique viral vectors and indicate potentially discrete roles of DC subtypes in activating the immune response following immunization with vectors that have distinct replication mechanisms.

A Review of Human Coronaviruses' Receptors: The Host-Cell Targets for the Crown Bearing Viruses.

A novel human coronavirus prompted considerable worry at the end of the year 2019. Now, it represents a significant global health and economic burden. The newly emerged coronavirus disease caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the primary reason for the COVID-19 global pandemic. According to recent global figures, COVID-19 has caused approximately 243.3 million illnesses and 4.9 million deaths. Several human cell receptors are involved in the virus identification of the host cells and entering them. Hence, understanding how the virus binds to host-cell receptors is crucial for developing antiviral treatments and vaccines. The current work aimed to determine the multiple host-cell receptors that bind with SARS-CoV-2 and other human coronaviruses for the purpose of cell entry. Extensive research is needed using neutralizing antibodies, natural chemicals, and therapeutic peptides to target those host-cell receptors in extremely susceptible individuals. More research is needed to map SARS-CoV-2 cell entry pathways in order to identify potential viral inhibitors.

Modular Lipoprotein Toxins Transferred by Outer Membrane Exchange Target Discrete Cell Entry Pathways.

ABSTRACTBacteria compete against related individuals by delivering toxins. In myxobacteria, a key delivery and kin discrimination mechanism is called outer membrane (OM) exchange (OME). Here, cells that display compatible polymorphic cell surface receptors recognize one another and bidirectionally transfer OM content. Included in the cargo is a suite of polymorphic SitA lipoprotein toxins. Consequently, OME between compatible cells that are not clonemates results in intoxication, while exchange between clonemates is harmonious because cells express a cognate repertoire of immunity proteins, which themselves are not transferred. SitA toxins belong to six nonhomologous families classified by sequence conservation within their N-terminal “escort domains” (EDs), while their C termini contain polymorphic nucleases that target the cytoplasmic compartment. To investigate how toxins delivered to the OM by OME translocate to the cytoplasm, we selected transposon mutants resistant to each family. Our screens identified eight genes that conferred resistance in a SitA family-specific manner. Most of these genes are predicted to localize to the cell envelope, and some resemble proteins that colicins exploit to gain cell entry. By constructing functional chimeric SitAs between families, we show that the ED determines the specificity of resistance. Importantly, a mutant that confers resistance to all six SitA families was discovered. This gene was named traC and plays an accessory role with traAB in OME. This work thus provides insight into the mechanism of kin discrimination in myxobacteria and provides working models for how SitA toxins exploit host proteins to gain cytoplasmic entry.

Methodological notes onpandemic virus SARS-CoV-2 research.

In the fight against the COVID-19 pandemic, many brilliant results have been achieved, but the thermodynamics of the novel SARS-CoV-2 coronavirus has been completely neglected. This is a serious systematic error, which can compromise the results of the entire pandemic virus SARS-CoV-2 research. In the present work, we therefore study the thermodynamics of SARS-CoV-2 in its environment, from air to endosome and endosome-independent cell entry pathways. In the study of the thermodynamics of the new coronavirus SARS-CoV-2 in air, the presence of pollen, bacteria, other viruses, spores, dust, but more particularly, that of nanoparticles of health interest at the same scale threshold as the spike proteins of the pandemic virus, such as particulate matter, cannot be neglected. This work therefore starts from a comparative study of the air environments in China and Italy, the first countries affected by the infection. Currently, a correlation between the spread of infection and pollution is still very controversial. But our paper is not concerned with this. We propose some methodological notes which lead us to the formulation of a general mathematical apparatus (an energy landscape theory), suitable to explain at the molecular level the energetic configurations of the quasi-species of the pandemic virus SARS-CoV-2 in its environment. We focus on complexes between the viral particle and other objects in its environment at the scale threshold of the spikes of the viral particle. Then, we wondered if such complexes can lead to the generation of more aggressive viral variants and how to predict their populations and energy configurations, in order to plan an adequate prophylaxis.